U.S. patent application number 10/769296 was filed with the patent office on 2005-02-03 for methods, devices and systems for characterizing proteins.
This patent application is currently assigned to Caliper Life Sciences, Inc.. Invention is credited to Chow, Andrea W., Fathollahi, Bahram, Mikkelsen, James C. JR., Spaid, Michael A., Winoto, Adrian.
Application Number | 20050027111 10/769296 |
Document ID | / |
Family ID | 34837805 |
Filed Date | 2005-02-03 |
United States Patent
Application |
20050027111 |
Kind Code |
A1 |
Chow, Andrea W. ; et
al. |
February 3, 2005 |
Methods, devices and systems for characterizing proteins
Abstract
A method of characterizing a polypeptide, comprising providing a
first capillary channel having a separation buffer disposed within,
wherein the separation buffer comprises a non-crosslinked polymer
solution, a buffering agent, a detergent, and a lipophilic dye. The
separation buffer is provided such that, at the time of detection,
the detergent concentration in the buffer is not above the critical
micelle concentration. The polypeptide is introduced into one end
of the capillary channel. An electric field is applied across a
length of the capillary channel, which transports polypeptides of
different sizes through the polymer solution at different rates.
The polypeptide is then detected as it passes a point along the
length of the capillary channel.
Inventors: |
Chow, Andrea W.; (Los Altos,
CA) ; Fathollahi, Bahram; (Palo Alto, CA) ;
Mikkelsen, James C. JR.; (Los Altos, CA) ; Spaid,
Michael A.; (Mountain View, CA) ; Winoto, Adrian;
(Mountain View, CA) |
Correspondence
Address: |
CALIPER LIFE SCIENCES, INC.
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043-2234
US
|
Assignee: |
Caliper Life Sciences, Inc.
Mountain View
CA
94043-2234
|
Family ID: |
34837805 |
Appl. No.: |
10/769296 |
Filed: |
January 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10769296 |
Jan 30, 2004 |
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10233760 |
Sep 3, 2002 |
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10233760 |
Sep 3, 2002 |
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09496849 |
Feb 2, 2000 |
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6475364 |
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09496849 |
Feb 2, 2000 |
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09243149 |
Feb 2, 1999 |
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Current U.S.
Class: |
530/412 |
Current CPC
Class: |
G01N 27/44791 20130101;
G01N 27/44747 20130101; C07K 1/24 20130101 |
Class at
Publication: |
530/412 |
International
Class: |
C07K 001/10 |
Claims
What is claimed is:
1. A method of separating two or more polypeptides, comprising:
providing a microfluidic device that has a body having at least a
first channel disposed therein, the first channel comprising first
and second channel segments, the first channel segment comprising a
separation buffer having a detergent disposed therein at a
concentration which is compatible with separation of the two or
more polypeptides; flowing the first sample material through the
first channel segment to separate the two or more polypeptides;
flowing the first sample material from the first channel segment
into the second channel segment; and introducing a first diluent
into the second channel segment, the diluent diluting the detergent
concentration in the separation buffer to a concentration which is
compatible with detection of the two or more polypeptides at a
detection region along the length of the second channel
segment.
2. The method of claim 1, wherein the separation operation
comprises an electrophoretic polypeptide separation, and detergent
concentration in the first channel segment is at or above a
critical micelle concentration (CMC) for the detergent.
3. The method of claim 2, wherein the detergent concentration in
the first channel segment is between about 0.1% and 1%.
4. The method of claim 2, wherein the detection comprises detection
of a lipophilic dye associated with polypeptides separated in the
first operation, and the detergent concentration in the second
channel segment is below the CMC for the detergent.
5. The method of claim 4, wherein the detergent concentration in
the second channel segment is between about 0.15% and 0.25%
6. The method of claim 4, wherein the detergent concentration in
the second channel segment is between about 0.05% and 0.4%.
7. The method of claim 4, wherein the detergent concentration in
the second channel segment is between about 0.1% and 0.3%.
8. The method of claim 1, wherein the diluent in the second channel
segment dilutes the separation buffer in a range of between about
1:2 to 1:30.
9. The method of claim 1, wherein the separation buffer comprising
a polymer matrix, a buffering agent, a first detergent and a
lipophilic dye.
10. The method of claim 9, wherein the separation matrix comprises
a non-crosslinked polymer solution.
11. The method of claim 10, wherein the non-crosslinked polymer
solution comprises a linear dimethylacrylamide polymer
solution.
12. The method of claim 11, wherein the linear polyacrylamide
polymer is present in the separation buffer at a concentration of
between about 0.1 and about 20% (w/v).
13. The method of claim 9, wherein the first detergent comprises an
alkylsulfonate detergent.
14. The method of claim 9, wherein the first detergent is selected
from sodium octadecylsulfate, sodium decylsulfate and sodium
dodecyl sulfate (SDS).
15. The method of claim 9, wherein the first detergent comprises
sodium dodecyl sulfate (SDS).
16. The method of claim 9, wherein the first detergent is present
in the separation buffer in the first channel segment at a
concentration of between about 0.01% to 1%.
17. The method of claim 9, wherein the buffering agent comprises
Tris-Tricine.
18. The method of claim 9, wherein the buffering agent is present
in the separation buffer in the first channel segment at a
concentration of between about 10 mM and about 200 mM.
19. The method of claim 9, wherein the lipophilic dye is a
fluorescent lipophilic dye.
20. The method of claim 89, wherein the lipophilic dye is present
in the separation buffer in the first channel segment at a
concentration of from about 0.1 .mu.M to about 1 mM.
21. The method of claim 1, wherein the diluent in the second
channel segment dilutes the separation buffer in a range of between
about 1:2 to 1:10.
22. The method of claim 1, wherein the diluent in the second
channel segment dilutes the separation buffer in a range of between
about 1:3 to 1:8.
23. The method of claim 1, wherein the diluent in the second
channel segment dilutes the separation buffer in a range of between
about 1:4 to 1:7.
24. A device for separating polypeptides, comprising: a body
structure having at least a first separation channel disposed
therein; and a separation buffer disposed in the first separation
channel, the separation buffer comprising: a non-crosslinked
polymer solution; a buffering agent having a concentration of
between about 10 mM and 200 mM; a first detergent having a
concentration of between about 0.01% and 1% (w/v); and a lipophilic
dye capable of binding to the polypeptide or polypeptides, the dye
having a concentration of between about 0..mu.M and 1 mM; and
25. The device of claim 24, wherein the detergent concentration in
the separation buffer is between about 0.05% and 0.4%.
26. The device of claim 24, wherein the detergent concentration is
between about 0.1% and 0.3%.
27. The device of claim 24, wherein the detergent concentration is
between about 0.15% and 0.25%.
28. The device of claim 24, further comprising a source of a second
detergent fluidly coupled to the separation channel which has a
concentration that is between about 0.05.times. and 3.times. a
concentration of the first detergent in the separation buffer.
29. The device of claim 24, further comprising a source of a second
detergent fluidly coupled to the separation channel which has a
concentration that is less than a concentration of the first
detergent in the separation buffer.
30. The device of claim 29, wherein the second detergent has a
concentration that is between about 0.0025% and 0.01% (w/v).
31. The device of claim 29, wherein the second detergent has a
concentration that is between about 0.0025% and 0.5% (w/v).
32. The device of claim 24, wherein the body structure comprises a
capillary element having the first capillary channel disposed
therein which is fluidly coupled to the separation channel.
33. The device of claim 24, further comprising a source of an ionic
solution fluidly coupled to the separation channel.
34. The device of claim 33, wherein the source of an ionic solution
comprises a salt selected from the group comprising NaCl, TrisCl,
and PBS.
35. The device of claim 34, wherein the ionic concentration of the
ionic solution is between about '100 mM and 500 mM.
36. The device of claim 34, wherein the ionic solution is disposed
in a reservoir of the body structure which is fluidly coupled to
the separation channel.
37. The device of claim 24, further comprising a source of at least
one standard protein ladder fluidly coupled to the separation
channel.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 10/233,760, filed on Sep. 3, 2002, which is a
divisional of U.S. patent application Ser. No. 09/496,849, filed on
Feb. 2, 2000 and issued on Nov. 5, 2002 as U.S. Pat. No. 6,475,364,
which is a continuation-in-part of U.S. patent application Ser. No.
09/243,149, filed on Feb. 2, 1999, now abandoned, the full
disclosure of which is hereby incorporated herein by reference in
its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] The characterization of biological compounds is an inherent
necessity of any endeavor that seeks to understand life, the
processes that sustain life, and the events and elements that
affect those processes. Typically, the understanding of life's
processes, and efforts at their control, focuses first at the basic
building blocks of life, namely the macromolecular compounds and
complexes that differentiate living organisms from mere lifeless
primordial ooze. Of particular interest in the understanding and
control of life processes are the nucleic acids and the proteins
they encode.
[0003] In the case of proteins, many characterization methods have
remained largely unchanged for decades. For example, current
protein characterization methods typically rely, at least in part,
upon sodium dodecylsulfate polyacrylamide gel electrophoresis, or
SDS-PAGE, to characterize proteins by their relative molecular
weights. These methods employ a slab or sheet of cross-linked
polyacrylamide. Proteins to be separated and characterized are
mixed with a detergent buffer (SDS) and are placed at one edge of
the slab, typically in a well. An electric field is applied across
the slab, drawing the highly charged detergent micelle containing
the proteins through the gel. Larger proteins move through the slab
gel more slowly than the smaller proteins, thereby separating out
from the greater micelle. After the separation, the gel is
contacted with a stain, typically "coomassie blue" or a silver
complexing agent, which binds to the different proteins in the gel.
In the case of coomassie blue stained gels, the slab gel must be
destained to remove the excess stain. These processes result in a
ladder of different proteins in the slab gel, separated by size.
Silver staining methods are similarly time consuming, and generally
yield qualitatively, although non-quantitatively stained gels.
Improvements to these processes have produced smaller gels that are
faster to run, gels that are purchased "ready-to-use," and
alternate staining processes. However, the basic SDS-PAGE process
has remained largely unchanged as a method of protein
characterization.
[0004] A number of attempts have been made to apply advances made
in other areas to protein characterization. For example, capillary
electrophoresis methods, which have proven successful in the
analysis of nucleic acids have been attempted in the
characterization of proteins. While these methods have proven
capable at separating proteins, differences in available labeling
chemistries, as well as fundamental structural and chemical
differences between proteins and nucleic acids have created
substantial barriers to the wide spread use of CE methods in
protein characterization. In particular, detection of separated
proteins traveling through a capillary has typically required the
covalent attachment of a labeling group to all of the proteins,
using relatively complex chemistry. Further, the presence of SDS in
protein separations, which ensures size based separations, creates
further difficulties in both labeling and separation within
capillary systems.
[0005] It would be desirable to provide methods, devices, systems
and kits for characterizing proteins and polypeptides, which would
have enhanced throughput, sensitivity and lower space, time and
reagent requirements. The present invention meets these and a
variety of other needs.
SUMMARY OF THE INVENTION
[0006] In one aspect, the present invention provides methods of
performing an analytical operation on a fluid first sample
material. The methods typically comprise providing a microfluidic
device that has a body having at least a first channel disposed
therein. The first channel comprises first and second channel
segments, where the first channel segment comprises a first fluid
environment compatible with the performance of a first operation.
The first sample material is flowed through the first channel
segment to perform the first operation. It is then flowed from the
first channel segment into the second channel segment. A first
diluent is flowed into the second channel segment, whereby the
diluent produces a second fluid environment within the second
channel segment, the second environment being more compatible than
the first environment with the second operation.
[0007] In a related aspect, the invention provides devices for
performing analytical operations on sample materials. The devices
generally comprise a body structure having a first channel segment
disposed within an interior portion of the body, the first channel
segment containing a first environment. The device also includes a
second channel segment disposed in the body and fluidly connected
to the first channel segment. At least a first diluent source is
also provided fluidly coupled to the second channel segment. The
devices also typically include a flow controller operably coupled
to the first diluent source for delivering the first diluent into
the second channel segment to provide a second environment within
the second channel segment.
[0008] In another aspect, the present invention provides a method
of characterizing a polypeptide, comprising providing a first
capillary channel having a separation buffer disposed within. The
separation buffer comprises a polymer matrix, a buffering agent, a
detergent, and a lipophilic dye. The polypeptide is introduced into
one end of the capillary channel. An electric field is applied
across a length of the capillary channel which transports
polypeptides of different sizes through the polymer matrix at
different rates. The polypeptide is then detected as it passes a
point along the length of the capillary channel.
[0009] Another aspect of the present invention is a device for
separating polypeptides. The device is comprised of a body
structure having at least a first capillary channel containing
separation buffer within. The separation buffer is comprised of a
polymer matrix, a buffering agent, a detergent, and a lipophilic
dye capable of binding to the polypeptide or polypeptides. A port
disposed in the body structure is in fluid communication with the
first capillary channel in order to introduce polypeptides into the
first capillary channel.
[0010] A further aspect of the present invention is a kit for use
in characterizing a polypeptide. The kit is comprised of a
microfluidic device hat comprises the elements of the devices
described above. The separation buffer is comprised of a polymer
matrix, a buffering agent, and a lipophilic dye. Each packaging
contains the body structure, the separation buffer, and the
lipophilic dye.
[0011] Another aspect of the present invention is a system for
characterizing a polypeptide. The system includes a body structure
having at least a first capillary channel containing a separation
buffer disposed therein. The separation buffer is comprised of a
polymer matrix, a buffering agent, a detergent, and a lipophilic
dye. An electrical power source is operably coupled to opposite
ends of the first capillary channel in order to apply an electric
field across a length of the capillary channel. A detector is
disposed in sensory communication with the capillary channel at a
first point to detect the polypeptide as it passes the first
point.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 illustrates a microfluidic device for use in
conjunction with the present invention.
[0013] FIG. 2 illustrates an overall system for use in
characterizing polypeptides according to the present invention.
[0014] FIG. 3 illustrates a plot of fluorescence intensity versus
detergent concentration for determining the critical micellar
concentration of the detergent in the given buffer.
[0015] FIG. 4 illustrates a chromatogram of a protein separation
performed in a microfluidic device using the methods of the
invention. The chromatogram is displayed as an emulated gel,
showing 12 separate separations, each as a separate lane of the
emulated gel.
[0016] FIG. 5 is a plot of the log of the molecular weight of the
standard proteins, separated as shown in FIG. 4, versus migration
time.
[0017] FIG. 6 is a chromatogram of molecular weight standards
showing the detergent-dye front peak.
[0018] FIG. 7 is a schematic illustration of a microfluidic device
for performing a post separation treatment in accordance with the
methods described herein.
[0019] FIG. 8 (A-D) shows plots of separation data illustrating the
effects of post separation dilution.
[0020] FIG. 9 is a schematic representation of a system for
characterizing polypeptides in accordance with the present
invention.
[0021] FIG. 10 is a schematic illustration of a microfluidic device
connected to an external capillary for performing a post separation
treatment in accordance with the methods described herein.
[0022] FIGS. 11A and 11B are a schematic representations of the
flow patterns within an intersection of a microfluidic device
performing protein analyses in accordance with the invention.
[0023] FIGS. 12A, 12B, and 12C are schematic illustrations of the
data produced in sequential analyses of a protein ladder, the same
data corrected using a first method, and the same data corrected
using a second method respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0024] I. Methods, Devices and Reagents
[0025] A. Generally
[0026] The present invention provides methods, devices, systems and
kits for use in characterizing polypeptides, proteins and fragments
thereof (collectively referred to herein as "polypeptides"). The
methods, devices, systems and kits of the invention are
particularly useful in characterizing polypeptides by their
molecular weight through electrophoretic migration of the
polypeptides through a polymer separation matrix that is contained
within a capillary channel, also referred to in general terms as
"capillary electrophoresis."
[0027] As noted previously, attempts have been made to separate
proteins and polypeptides using capillary electrophoresis methods.
Because capillary electrophoresis uses a closed system, e.g., a
capillary, labeling of the proteins has typically been carried out
prior to the separation. This has generally taken the form of
covalent attachment of labeling groups to all of the proteins in
the mixture to be separated. Once separated, the label upon each
protein can then be detected. Covalent labeling techniques often
involve complex chemistries, and at the very least, require
additional steps in advance of separating the proteins.
Additionally, labels are generally relatively large structures
which may adversely affect the determination of a protein's
molecular weight. While some have attempted to use non-covalent,
associative dyes, such attempts have generally provided less than
acceptable results.
[0028] In accordance with at least a first aspect of the present
invention, however, methods are provided for characterizing and/or
separating proteins by capillary electrophoretic methods, which are
rapid, reproducible, and do not involve complex sample preparation
steps prior to performing the separation. In particular, the
methods of the present invention provide a first capillary channel
that includes a separation buffer disposed therein, where the
separation buffer includes a polymer matrix, a buffering agent, a
detergent and a lipophilic dye. In accordance with preferred
aspects of the invention, the detergent and buffering agent are
present within the separation buffer at concentrations that are at
or below the critical micelle concentration ("CMC"). By maintaining
the detergent and buffer concentrations at or below the CMC,
adverse effects, such as dye binding to detergent micelles can be
minimized. Without being bound to a particular theory of operation,
it is believed that dye binding to detergent micelles within a
capillary system in previously described systems, has resulted in
substantial background signal and has yielded signal irregularities
during a separation, e.g., bumps and dips in a signal baseline. The
methods of the present invention, on the other hand, carefully
control the various components of the system to avoid or at least
minimize these adverse effects. In particularly preferred aspects,
the buffer and detergent are provided at a level at or below the
CMC at least at the point at which the separated components of the
operation are to be detected, thereby avoiding the dye binding to
the micelles that gives higher background signals. This can be a
result of the overall system being maintained and/or run at levels
below the CMC, e.g., buffer and detergent concentrations, or it can
be a result of an in situ treatment of the sample, buffer,
detergent fluids, e.g., dilution, reagent addition or other
solution modification, which reduces the separation buffer in the
detected portion of the system to a level below the CMC.
[0029] In practice, the protein or polypeptide sample that is to be
analyzed and or characterized, is typically pretreated to denature
the protein and provide adequate coating of the protein by the
detergent, as well as provide adequate labeling of the coated
proteins in the sample.
[0030] The protein or polypeptide that is to be characterized (or
mixture of polypeptides that are to be separated) are then
introduced into the capillary channel, typically at one end of a
channel segment. By applying an electric field across the length of
the capillary channel, polypeptides of different size will migrate
through the polymer solution at different rates. The polypeptides,
which are coated in detergent that has a substantial charge
associated with it, will migrate in one direction through the
capillary channel. Polypeptides of different molecular weights,
however, will migrate through the polymer solution at different
rates, and will be separated out. While traveling through the
separation buffer in the channel, the polypeptides will pick up the
lipophilic dye that is present within the separation buffer, as
well as bringing any associated dye which was optionally included
with the sample, e.g., during sample pretreatment, dilution or the
like.
[0031] In the context of the separation, once separated from each
other, the polypeptides, which at this point have a level of an
associative lipophilic dye associated with them, can be detected by
virtue of that dye, at a point in the capillary channel downstream
of the point at which they were introduced.
[0032] B. Sample Pretreatment
[0033] As noted above, prior to their characterization, protein or
polypeptide containing samples are typically pretreated with an
appropriate detergent containing buffer. In particularly preferred
aspects, the polypeptide sample mixture is pretreated in a buffer
that comprises the same buffering agent as the separation buffer
and the same detergent that is used in the separation buffer, in
order to ensure denaturation of the protein prior to its
separation. Denaturation of the protein ensures a linear molecule
during separation, so that the separation profile of a protein is
more closely related to its molecular weight, regardless of whether
the native protein is globular, linear, filamentous, or has some
other conformation. Pretreatment is typically carried out in the
presence of detergent at a concentration that is greater than the
protein concentration of the sample (w/v), and preferably greater
than about 1.4.times. of the protein concentration (w/v) in the
sample.
[0034] In order to avoid interfering effects of detergent bound
dye, it is often desirable to perform sample pretreatment in a
detergent concentration that is less than or approximately equal to
the concentration of detergent in the running buffer, from about
0.05.times. to about 3.times., of the detergent concentration of
the running buffer.
[0035] In preferred aspects, the concentration of SDS in the
pretreatment buffer is less than that used in the running buffer.
Thus, the sample pretreatment is typically carried out in the
presence of a detergent concentration of between about 0.05% and
2%, preferably, between about 0.05% and about 1% and more
preferably, less than about 0.5%. If the sample material is then
diluted in the loaded sample, e.g., from about a 1:2 to about a
1:20 dilution, this results in a detergent level in the loaded
sample of between about 0.0025% to about 1% detergent, preferably,
from about 0.0025% to 0.5%, and again, more preferably less than
about 0.5%.
[0036] These levels are in contrast to conventional SDS-PAGE
separations where samples are pretreated in detergent
concentrations that can be upwards of 5 to 20 times that of the
separation buffer. In particular, sample pretreatment for typical
SDS-PAGE methods is generally carried out in loading buffers that
have detergent, e.g., SDS, concentrations of 2% or greater (See,
e.g., U.S. Pat. No. 5,616,502) in 50 mM buffer, while the running
buffer contains only 0.1% detergent. Use of these relatively high
detergent levels in the loading buffer as compared to the running
buffer when used in capillary systems as described herein however,
gives rise to a much larger interfering detergent front that tends
to co-elute with polypeptides having molecular weights in a
desirable range. For example, FIG. 6 shows a chromatogram of a set
of molecular weight standards (see Examples section, below). In the
example shown, the peak associated with the detergent front eluted
at approximately 43 seconds, which would correspond to the elution
time for proteins or polypeptides having molecular weights in the
range of 60 to 70 kD, an important molecular weight range in
protein analyses.
[0037] By reducing the concentration of detergent in the sample
pretreatment step, any interfering peak is also reduced. This has
proven effective despite the previously held belief in the art that
sample pretreatment required high levels of detergent, e.g., 2% or
higher. Further, controlling the ionic strength and detergent
concentration of the sample pretreatment and separation buffers in
accordance with the parameters set forth herein, allows one to
somewhat control the elution profile of the detergent front, e.g.,
causing its elution before or after the polypeptides that are to be
characterized.
[0038] Also in preferred aspects, the detergent used in
pretreatment is the same detergent used in the separation buffer,
e.g., SDS. Generally, pretreatment conditions can be varied
depending upon the conditions of the overall separation, e.g., the
nature of the proteins to be separated, the medium in which the
samples are disposed, e.g., buffer and salt concentrations, and the
like, as described for the separation buffers, below. In
particular, SDS and salt concentrations may be varied, e.g., within
the parameters set forth herein, so as to optimize for a given
separation.
[0039] C. Separation Buffers
[0040] In accordance with the present invention, a separation
buffer is used in carrying out the methods described herein, which
buffer comprises a polymer matrix, a buffering agent, a detergent
and a lipophilic dye. A variety of polymer matrices can be used in
accordance with the present invention, including cross-linked
and/or gellable polymers. However, in preferred aspects,
non-crosslinked polymer solutions are used as the polymer matrix.
Non-crosslinked polymer solutions that are suitable for use in the
presently described methods have been previously described for use
in separation of nucleic acids by capillary electrophoresis, see
e.g., U.S. Pat. Nos. 5,264,101, 5,552,028, 5,567,292, and
5,948,227, each of which is hereby incorporated herein by
reference. Such non-crosslinked or "linear" polymers provide
advantages of ease of use over crosslinked or gelled polymers. In
particular, such polymer solutions, because of their liquid nature,
are more easily introduced into capillary channels and are ready to
be used, whereas gelled polymers typically require a cross-linking
reaction to occur while the polymer is within the capillary.
[0041] Generally, the most commonly utilized non-crosslinked
polymer solution comprises a polyacrylamide polymer, which
preferably is a polydimethylacrylamide polymer solution which may
be neutral, positively charged or negatively charged. In
particularly preferred aspects, a negatively charged
polydimethylacrylamide polymer is used, e.g.,
polydimethylacrylamide-co-acrylic acid (See, e.g., U.S. Pat. No.
5,948,227). Surprisingly, the use of polydimethylacrylamide polymer
solutions does not result in any smearing of the
proteins/polypeptides that are being separated in a capillary
system. Without being bound to a particular theory of operation, it
is believed that the polymer solutions have a dual function in the
systems described herein. The first function is to provide a
matrix, which retards the mobility of larger species moving through
it relative to smaller species. The second function of these
polymer solutions is to reduce or eliminate electroosmotic flow of
the materials within a capillary channel. It is believed that the
polymer solutions do this by adsorbing to the capillary surface,
thereby blocking the sheath flow, which characterizes
electroosmotic flow.
[0042] Typically, the non-crosslinked polymer is present within the
separation buffer at a concentration of between about 0.01% and
about 30% (w/v). Of course different polymer concentrations may be
used depending upon the type of separation that is to be performed,
e.g., the nature and/or size of the polypeptides to be
characterized, the size of the capillary channel in which the
separation is being carried out, and the like. In preferred
aspects, for separation of most polypeptides, the polymer is
present in the separation buffer at a concentration of from about
0.01% to about 20% and more preferably, between about 0.01% and
about 10%.
[0043] The average molecular weight of the polymer within the
polymer solutions may vary somewhat depending upon the application
for which the polymer solution is desired. For example,
applications that require higher resolution may utilize higher
molecular weight polymer solutions, while less stringent
applications can utilize lower molecular weight polymer solutions.
Typically, the polymer solutions used in accordance with the
present invention have an average molecular weight in the range of
from about 1 kD to about 6,000 kD, preferably between about 1 kD
and about 1000 kD, and more preferably, between about 100 kD and
about 1000 kD.
[0044] In addition to the percent charge and molecular weights
described above, the polymers used in accordance with the present
invention are also characterized by their viscosity. In particular,
the polymer components of the system described herein typically
have a solution viscosity as used within the capillary channel, in
the range of from about 2 to about 1000 centipoise, preferably,
from about 2 to about 200 centipoise and more preferably, from
about 5 to about 100 centipoise.
[0045] In addition to incorporation of a non-crosslinked polymer
solution, the separation buffers used in practicing the present
invention also comprise a buffering agent, a detergent, and a
lipophilic dye.
[0046] As noted previously, polypeptides typically vary a great
deal in their physicochemical properties, and particularly in their
charge to mass ratios, depending upon their amino acid composition.
As such, different polypeptides will generally have different
electrophoretic mobilities under an applied electric field. As
such, electrophoretic separation of proteins and other polypeptides
typically utilizes a detergent within the running buffer, in order
to ensure that all of the proteins/polypeptides migrate in the same
direction under the electric field. For example, in typical protein
separations, e.g., SDS-PAGE, a detergent (sodium dodecylsulfate or
SDS) is included in the sample buffer. The proteins/polypeptides in
the sample are coated by the detergent which to provide the various
proteins/polypeptides with a substantial negative charge. The
negatively charged proteins/polypeptides then migrate toward the
cathode under an electric current. In the presence of a sieving
matrix, however, larger proteins will move more slowly than smaller
proteins, thereby allowing for their separation.
[0047] In accordance with certain aspects of the invention, each of
the detergent, buffering agent and dye components of the separation
buffer is selected and provided at a concentration so as to
minimize any adverse interactions among them, which interactions
can interfere with the separation and characterization of proteins
or polypeptides, e.g., reduce separation efficiency, signal
sensitivity, production of aberrant signals, or the like. In
particular, the buffering agent and detergent are typically
provided at concentrations which optimize separation efficiencies
of polypeptides, but which minimize background signal, and baseline
signal irregularities. As noted previously, it has been observed
that dye binding to detergent micelles produces a substantial level
of background signal during capillary separations, as well as
giving rise to various baseline irregularities, e.g., bumps and
dips.
[0048] Accordingly, in a first aspect, polypeptide separation
and/or characterization is accomplished by providing the buffering
agent and the detergent at concentrations which are below the point
at which the detergent begins to form excessive independent
micelles, to which dye may bind, within the buffer solution.
Typically, the concentration at which micelles begin to form is
termed the critical micelle concentration ("CMC"). Restated, the
CMC is the highest monomeric detergent concentration obtainable and
thus, the highest detergent potential obtainable. Helenius et al.,
Methods in Enzymol. 56(63):734-749 (1979).
[0049] The CMC of a detergent solution decreases with increasing
size of the apolar moiety (or hydrocarbon tail), and to a lesser
extent, with the decreasing size and polarity of the polar groups.
Helenius et al., supra. Thus, whether a detergent solution is above
or below its CMC is determined not only by the concentration of the
detergent, but also by the concentration of other components of the
solution which can have an effect on the CMC, namely the buffering
agent and ionic strength of the overall solution. Accordingly, in
the methods, systems and devices of the present invention, the
separation buffer is provided with a detergent concentration and a
concentration of buffering agent, such that the separation buffer
is maintained at or below the CMC.
[0050] A number of methods can be used to determine whether a
buffer is below its CMC. For example, Rui et al., Anal. Biochem.
152:250-255 (1986) describes the use of a fluorescent
N-phenyl-1-naphthylamine dye to determine the CMC of detergent
solutions. In the context of the separation buffers described
herein, the detergent is typically provided at a concentration that
is at or below the CMC for the separation buffer. In particularly
preferred aspects, the detergent concentration is at or just below
the CMC for the buffer. Determination of optimal concentration of
detergent may be determined experimentally. In particular, using
the lipophilic dyes described herein, one can measure the relative
micelle concentration in a detergent solution by measuring the
fluorescence of the solution as a function of detergent
concentration. For example, FIG. 3 illustrates a plot of
fluorescent intensity of SDS solutions containing 10 .mu.M of a
fluorescent lipophilic dye (Syto 61, Molecular Probes Inc.) as a
function of SDS concentration. The critical micellar concentration
is indicated by the steep increase in the fluorescent intensity,
indicated as point A. In accordance with the present invention,
therefore, where it is indicated that the detergent concentration
is at or below the CMC, it is understood that the detergent
concentration will be a concentration that falls either on or below
the steep portion of a plot like that shown, and particularly,
below the point on the curve indicated as point B, and preferably,
within or below the region marked as point A.
[0051] As noted, the CMC of a detergent varies from one detergent
to another, and also varies with the ionic strength of the buffer
in which the detergent is disposed. In typical separation
operations and buffers, the detergent concentration in the
separation buffer is provided at a concentration above about 0.01%
(w/v), but lower than about 0.5%, while the buffering agent is
typically provided at a concentration of from about 10 mM to about
500 mM, provided that the buffer is maintained at or below the
CMC.
[0052] Detergents incorporated into the separation buffer can be
selected from any of a number of detergents that have been
described for use in electrophoretic separations. Typically,
anionic detergents are used. Alkyl sulfate and alkyl sulfonate
detergents are generally preferred, such as sodium
octadecylsulfate, sodium dodecylsulfate (SDS) and sodium
decylsulfate. In particularly preferred aspects, the detergent
comprises SDS. In SDS embodiments, the detergent concentration is
generally maintained at concentrations described above. In
preferred aspects, SDS concentrations in the separation buffers are
therefore typically greater than 0.01% to ensure adequate coating
of the proteins in the sample, but less than about 0.5% to prevent
excessive micelle formation. In preferred aspects, the detergent
concentration is between about 0.02% and about 0.15%, and
preferably, between about 0.03% and 0.1%.
[0053] In buffers utilizing preferred detergent concentrations, the
buffering agent is typically selected from any of a number of
different buffering agents. For example, buffers that are generally
used in conjunction with SDS-PAGE applications are also
particularly useful in the present invention, such as tris,
tris-glycine, HEPES, CAPS, MES, Tricine, combinations of these, and
the like. In particularly preferred aspects, however, buffering
agents are selected that have very low ionic strengths. Use of such
buffers allows one to increase the concentration of detergent
without exceeding the CMC. Preferred buffers of this type include
zwitterionic buffers, such as amino acids like histidine and
Tricine, which have a relatively high buffering capacity at the
relevant pH, but which have extremely low ionic strengths, due to
their zwitterionic nature. Buffering agents that comprise
relatively large ions having relatively low mobilities within the
system are also preferred for their apparent ability to smooth out
the signal baseline, e.g., using Tris as a counterion.
[0054] In the case of the preferred detergent solutions, e.g., SDS,
sodium octadecylsulfate, sodium decylsulfate, and the like, at the
above-described concentrations, the buffering agent is typically
provided at concentrations between about 10 mM and about 200 mM,
and preferably at a concentration of between about 10 mM and about
100 mM. In particularly preferred aspects, Tris-Tricine is used as
the buffering agent at a concentration of between about 20 mM and
about 100 mM.
[0055] With reference to the foregoing discussion, it can be seen
that the most preferred separation buffer comprises SDS at a
concentration of between about 0.03% and about 0.1%, and
Tris-Tricine as the buffering agent, at a concentration of between
about 20 mM and about 100 mM, with each being provided such that
the buffer is at or below the CMC, when operating under the normal
operating conditions of the overall system/method.
[0056] In addition to the foregoing components, the separation
buffer also typically comprises an associative dye or other
detectable labeling group, which associates with the proteins and
polypeptides that are to be characterized/separated. This enables
the detection of proteins and/or polypeptides as they are traveling
through the separation buffer. As used herein, an "associative dye"
refers to a detectable labeling compound or moiety, which
associates with a class of molecules of interest, e.g., a protein
or peptide, preferentially with respect to other molecules in a
given mixture. In the case of protein or polypeptide
characterization, lipophilic dyes are particularly useful as
protein or polypeptide associative dyes.
[0057] Examples of particularly preferred lipophilic dyes for use
in the present invention include fluorescent dyes, e.g.,
merocyanine dyes, such as those described in U.S. Pat. No.
5,616,502, which is incorporated herein by reference. Particularly
preferred dyes include those that are generally commercially
available from Molecular Probes, Inc. (Eugene Oreg.) as the Sypro
Red.TM., Sypro Orange.TM., and Syto 61.TM. dyes. Such dyes are
generally intended for use in staining slab gels, in which one can
wash away excess dye, and eliminate any adverse effects of SDS in
the gel, e.g., through washing. However, surprisingly, it has been
discovered by the present inventors, that these dyes are
particularly useful in SDS capillary gel electrophoresis (SDS-CGE),
giving surprising sensitivity and with little or no "smearing" or
interference from the detergent, when the buffers are formulated as
described herein.
[0058] Further, and more unexpected than the compatibility of the
dyes with the separation buffer, is that the incorporation of the
lipophilic dye into the separation buffer within the capillary
channel does not create excessive background signal which would
reduce the sensitivity of the assay. In particular, by providing
the dye within the separation buffer one would expect to observe a
relatively high background signal from the dye that is in the
buffer. Accordingly, one would expect to be required to include the
dye within the sample solution, but not within the separation
buffer in the channel. However, this latter techniques results in
an extremely low signal level during separation. By including the
dye in the separation buffer within the capillary channel, signal
is maintained high while background is maintained surprisingly low.
The lipophilic dyes used in the present invention are generally
present within the separation buffer at concentrations between
about 0.1 .mu.M and 1 mM, more preferably, between about 1 .mu.M
and about 20 .mu.M.
[0059] D. Post-Separation Treatment
[0060] In contrast to the methods described above, wherein the
sample is pretreated and separated under buffer and detergent
concentrations that are optimized for the dye system utilized,
e.g., maintained below the CMC of the particular detergent, in
certain aspects, the buffer/detergent conditions in which the
sample components exist are altered after separation of those
components and during or immediately prior to detection of those
components, whereupon the adverse effects of detergent micelles are
reduced or eliminated. Specifically, sample components, e.g.,
polypeptides are separated under optimized separation buffer and
detergent conditions or concentrations that may be at, above or
below the CMC. Once the sample components are separated, these
conditions are altered such that the buffer and/or detergent
concentrations at the detection point are optimized for the
detection step, for example reducing those levels to a level below
the CMC. In particular, often, once the detergent level and/or
buffer concentrations are adjusted below the CMC, the micelles
disperse and the adverse effects of dye binding to micelles are
reduced or eliminated.
[0061] Typically, in the case of polypeptide separations, altering
the environment is carried out by adding one or more diluents into
the separated sample components prior to their passing the
detector, such that the sample-containing separation buffer is at
or below the CMC. This is optionally done by altering the ratio of
detergent and buffering agent to elevate the CMC to at or above the
operating concentration of detergent, and/or dilute the detergent
level such that it falls below the CMC. Thus, the diluent may add
to, maintain or reduce the concentration of buffering agent while
typically reducing the level of detergent, or it may maintain the
detergent concentration while reducing the concentration of
buffering agent. In either instance, the desired goal is to
eliminate detergent micelles at the point and time of detection. In
a similar fashion, materials may be added that effectively break up
detergent micelles, e.g., co-detergents.
[0062] Where post-separation treatment is used, the separation
buffer composition can span a wider range of buffer and detergent
concentrations. For example, the separation buffer typically
includes a buffering agent, e.g., as described above, at
concentrations from about 10 to about 200 mM, and detergent
concentrations of from about 0.01 to about 1.0%, and typically
above the CMC, e.g., above about 0.05% and preferably above about
0.1%. Detection of lipophilic dyes, on the other hand, is
preferably carried out in the absence of excessive detergent
micelles, which bind the dye and contribute to excessive background
signals. Thus, dilution of the separation buffer is typically
practiced to reduce the detergent concentration to a level below
the CMC of the detergent, e.g., less than about 0.1%. Accordingly,
the dilution step preferably dilutes the separation buffer from
about 1:2 to about 1:30 prior to detection. While this also dilutes
the sample components to be detected, the substantial reduction in
background as a result of the dilution enables easy detection at
very low levels of sample material.
[0063] In accordance with this aspect of the invention,
microfluidic devices are particularly well suited for carrying out
these methods. In particular, the inclusion of integrated fluid
channel networks permits the ready addition of diluents and other
reagents into flowing streams of materials. Specifically, diluent
channels are provided immediately upstream of the detection zone so
as to deliver diluent into the detection zone along with the
separated sample components. The sample components are then
detected in the absence of interfering detergent micelles. An
example of a particularly preferred channel layout for a
microfluidic device for accomplishing this post separation
treatment is shown in FIG. 7, and described in greater detail,
below. As used herein, the terms "upstream" and "downstream" refer
to the relative positioning of the element so described when
considered in the context of the direction of flow of the material
of interest, e.g., fluid, sample components, etc., during normal
operation of the system being described. Typically, the phrase
upstream refers to the direction toward the sample or buffer
reservoir connected to a particular channel, while downstream
refers to the direction of the waste reservoir connected to a
particular channel.
[0064] E. Capillary Channels and Devices
[0065] 1. Generally
[0066] The present invention also provides devices and systems for
use in carrying out the above described protein characterization
methods. The devices of the present invention typically include a
supporting substrate which includes a separation zone into which is
placed the separation buffer. A sample that is to be
separated/characterized is placed at one end of the separation zone
and an electric field is applied across the separation zone,
causing the electrophoretic separation of the proteins/polypeptides
within the sample. The separated proteins/polypeptides are then
separately detected by a detection system disposed adjacent to and
in sensory communication with the separation zone.
[0067] 2. Conventional Capillary Systems
[0068] In at least a first aspect, the methods of the present
invention are applicable to conventional capillary-based separation
systems. Accordingly, in these aspects, the supporting substrate
typically comprises a capillary tube, e.g., fused silica, glass or
polymeric capillary tube, which includes a capillary channel
disposed through it. At least a portion of the capillary channel in
the tube comprises the separation zone of the capillary. Separation
buffer is placed into the capillary channel by, e.g., pressure
pumping, capillary action or the like, and the sample to be
separated/characterized is injected into one end of the capillary
channel. One end of the capillary tube is then placed into fluid
contact with a cathode reservoir (having a cathode in contact with
the reservoir) at one end and with an anode reservoir (having an
anode in contact with the reservoir) at the other, and an electric
field is applied through the capillary tube to electrophorese the
sample material through the capillary tube and the contained
separation buffer. As the proteins and polypeptides travel through
the separation buffer they associate with the lipophilic dye which
is then detected toward the cathode end of the capillary channel by
the detection system.
[0069] In the case of a post separation treatment step, e.g., as
described above, additional buffer solutions are typically
introduced into the flow path of the sample components post
separation, by connecting additional flow paths or capillaries to
the main separation capillary, such that the separated components
exiting the separation capillary are mixed with the additional
buffers or diluents. A detection chamber or capillary is also
connected at this junction, such that all of the materials flow
into the detection zone to be detected.
[0070] 3. Microfluidic Devices
[0071] In particularly preferred aspects, the methods of the
invention are carried out in a microfluidic device that provides a
network of microscale capillary channels disposed within a single
integrated solid substrate. In particular, the supporting substrate
typically comprises an integrated body structure that includes a
network of one or more microscale channels disposed therein, at
least one of which is a separation channel. The separation buffer
is placed within at least the separation channel. In preferred
aspects, the microfluidic channel network comprises at least a
first separation channel that is intersected by at least a first
sample injection channel. The intersection of these two channels
forms what is termed an "injection cross." In operation, the sample
material is injected through the injection channel and across the
separation channel. The portion of the material within the
intersection is then injected into the separation channel whereupon
it is separated through the separation buffer. A detector is
disposed adjacent the separation channel to detect the separated
proteins.
[0072] In particularly preferred aspects, the microfluidic devices
used in accordance with the present invention comprise a plurality
of sample wells in fluid communication with a sample injection
channel which, in turn, is in fluid communication with the
separation channel. This allows he analysis of multiple different
samples within a single integrated microfluidic device. Examples of
particularly preferred microfluidic devices for use in accordance
with the present invention are shown and described in commonly
owned U.S. patent application Ser. No. 09/165,704, filed Oct. 2,
1998, which is incorporated herein by reference in its entirety for
all purposes. An example of such a microfluidic device is
illustrated in FIG. 1. As shown, the device 100, comprises a planar
body structure 102 which includes a plurality of interconnected
channels disposed within its interior, e.g., channels 104-138. A
number of reservoirs 140-170 are also disposed in the body
structure 202 and are in fluid communication with the various
channels 104-138. Samples to be analyzed and buffers are placed
into these reservoirs for introduction into the channels of the
device.
[0073] In operation, the separation buffer to be used in the
separation/characterization is first placed into one reservoir,
e.g., reservoir 166, and allowed to wick into all of the channels
of the device, thereby filling these channels with the separation
buffer. Samples that are to be separated/characterized are
separately placed into reservoirs 140-162. The separation buffer is
then placed into reservoirs 164, 168 and 170 and is already present
in reservoir 166. Through the application of appropriate electric
currents, the first sample material is transported or
electrophoresed from its reservoir, e.g., reservoir 140, to and
through the main injection intersection 172 for channel 104, via
channel 120 and 116. This is generally accomplished by applying the
current between reservoir 140 and 168. Low level pinching currents
are typically applied at the intersection in order to prevent
diffusion of the sample material at the intersection, e.g., by
supplying a low level of current from reservoirs 166 and 170 toward
reservoir 168 (see, e.g., WO 96/04547). After a short period of
time, the current is switched such that the material in the
intersection is electrophoresed down the main analysis channel 104,
e.g., by applying the current between reservoirs 170 and 166.
Typically, a slight current is applied after the injection to pull
material in channels 116 and 134 back from the intersection, to
avoid leakage into the separation channel. While the first sample
is being electrophoresed down the main channel 104, the next sample
to be analyzed is preloaded by electrophoresing the sample material
from its reservoir, e.g., reservoir 142, toward preload reservoir
164 through the preload intersection 174. This allows for only a
very short transit time to move the sample material from its
preloaded position to the injection intersection 172. Once the
first sample analysis is completed, the second sample material is
electrophoresed across the injection intersection 172 and injected
down the main analysis channel, as before. This process is repeated
for each of the samples loaded into the device.
[0074] A detection zone 176 is typically provided along the main
analysis channel 104, in order to provide a point at which signal
may be detected from the channel. Typically, the devices described
herein are fabricated from transparent materials. As such, the
detection window for optically detected analyses can be located at
virtually any point along the length of the analysis channel 104.
As the separated sample passes the detection window, the lipophilic
dye that is associated with the polypeptide fragments is detected.
The amount of time required for each polypeptide fragment to travel
through the separation channel then allows for the characterization
of the particular polypeptide, e.g., as a measure of its molecular
weight. In particular, the retention time of an unknown polypeptide
is compared to the retention time of known molecular weight
standards, and the approximate molecular weight of the unknown can
be thereby determined, e.g., interpolated or extrapolated from the
standards.
[0075] As noted previously, the post-separation treatment methods
described herein are particularly advantaged by the use of
microfluidic channel systems. Specifically, coupling of sources of
diluent to the main separation channel is a simple matter of
providing channels connected to that channel at the appropriate
location, e.g., at a point that falls after the separation has
occurred, but before the detection zone or window. An example of a
microfluidic channel network for accomplishing this is illustrated
in FIG. 7. As shown, the microfluidic device 700 includes a body
702 that includes a channel network disposed within its interior
portion. Typically, the device shown in FIG. 7 will be fabricated
in the same manner described above with reference to FIG. 1. The
channel network includes a main channel 704 that is in fluid
communication a plurality of different sample material reservoirs
706-722 and 728 via sample channels 706a-722a and 728a,
respectively. Preload/waste reservoir channel/reservoirs 724/724a
and 726/726a are also shown. The main channel 704 is connected to a
buffer reservoir 736 and a waste reservoir 732 and includes a
detection zone 738. As shown, two diluent channels 730a and 734a
are provided in communication with main channel 704, on opposite
sides of the main channel 704, at a point immediately upstream (in
the direction of operational flow of material) from the detection
zone, but downstream of the major portion of the main channel 704,
where the function of that channel, e.g., separation, occurs.
Diluent channels 730a and 734a are also in communication with
diluent sources, e.g., reservoirs 730 and 734, respectively, so as
to be able to deliver diluent from these sources to the main
channel 704.
[0076] In operation in a polypeptide separation, where one wishes
to characterize a sample, e.g., containing a polypeptide mixture,
one fills the channels of the device 700 with the separation
buffer. In the case of post separation treatment, this buffer need
not adhere to the strictures defined above, because the concern
over excessive micelle formation is largely lacking. Typically, in
these cases, the concentration of detergent is not as important as
in the pretreatment methods. In particular, the separation buffer
can have higher concentrations of detergent, e.g., from about 0.1%
to about 2.0%. Typically, the detergent concentration will be in
excess of 0.1%. Filling the channel networks is typically carried
out by depositing the separation buffer into one well, e.g., waste
reservoir 732. The separation buffer then wicks throughout the
channel network until it reaches each of the other reservoirs
706-730 and 734-736. Optionally, slight pressure is applied to the
waste reservoir 732 to expedite filling of the channel network. An
additional quantity of buffer, e.g., separation buffer, is placed
into buffer reservoir 736 and load/waste reservoirs 724 and 726. A
diluent material is placed into diluent reservoirs 730 and 734.
[0077] The sample material is placed into one or more of the sample
reservoirs 706-722, and 728. Optionally, a number of different
sample materials are placed into different reservoirs. The device
is then placed into a controller/detector apparatus, e.g., a 2100
Bioanalyzer from Agilent Technologies, which directs movement of
the sample materials through the channels of the device, e.g., by
controlled electrokinetic methods, as described in U.S. Pat. No.
5,976,336, which is incorporated herein by reference in its
entirety for all purposes. A sample placed into, e.g., reservoir
706 is moved along sample channel 706a until it crosses channel
704, and flowed toward load waste reservoir 726 via channel 726a.
The portion of the sample material at the intersection of the
sample loading channel 706a and the main channel 704 is then
injected into the separation channel 704, and moved therethrough.
Under an applied electric field, this portion of the sample that is
moving through the separation buffer separates into its constituent
elements as it moves along the channel 704. As it travels, the
sample components, and in some cases the detergent micelles, pick
up the lipophilic dye that is present in the separation buffer.
Diluent buffering agents containing a lower concentration or no
detergent is introduced in a continuous fashion into channel 704
via channels 730a and 734a. This diluent dilutes the separation
buffer to a point that is below the CMC for the detergent,
resulting in an elimination of excess detergent micelles. The
diluted sample constituents bearing the lipophilic dye are then
detected at the detection window 738. In some cases, fluidic
dilution is accomplished through the actual introduction of fluid
through the side channels. However, in preferred aspects, side
channels 730a and 734a typically contain the same separation matrix
present throughout the channel network. As such, dilution is
carried out by the electrophoretic introduction of the ionic
species from the buffering solution are introduced
electrophoretically into the separation channel, to effectively
dilute the species in the separation channel. In alternative
aspects, the side channels 730a and 734a are provided free of any
matrices, e.g., they can support pressure based or electroosmotic
flow, and bulk fluid is introduced into the main channel 704, to
dilute the separated sample components. As noted, the rate at which
diluent is added to the channel is selected to reduce the detergent
concentration in the channel at the detection point to a level
below about the CMC for the detergent under the particular
conditions. Typically, this comprises from about a 1:2 to about a
1:30 dilution of the detergent. Thus, in the case where the
separation buffer includes, e.g., 2% SDS in a 30 mM Tris Tricine
buffer, it is generally desirable to dilute the detergent level to
below about 0.1% and preferably to about 0.05% SDS. Thus, the
dilution is from about 2 to 3 fold to about 4 fold. Of course, as
noted previously, the CMC of a particular detergent can vary
depending upon the nature and concentration of the buffer.
[0078] Although described primarily in terms of diluting a
polypeptide separation buffer to a point that is below the CMC of
the detergent in that buffer, it will be appreciated that the
post-separation treatment methods described herein are more broadly
applicable. Specifically, such methods can be used in a variety of
analytical operations where a subsequent operation in a chain of
analytical method steps requires a different environment from the
immediately preceding step or operation, which environment can be
sufficiently altered by the addition of reagents, buffers, or
diluents, for that subsequent operation. The above-described
methods illustrate an example where the environment that is
optimized for separation of polypeptides may not be optimally
compatible with the optimized detection environment. Thus, in
accordance with the broadest understanding of this aspect of the
invention, the term "diluent refers to an added element, e.g.,
fluid, buffering agent, etc., that alters the environment into
which it is introduced. Alteration of an environment in this sense
includes changing physical properties of the environment, e.g., the
presence of detergent micelles, reducing the viscosity of a
solution, but also includes changing the chemical environment,
e.g., titrating a buffer to yield a change in he pH of a solution,
e.g., to yield a operable environment for a pH sensitive dye or
other labeling species, varying a salt concentration of a solution
to affect a change in hydrophobicity/hydrophilicity or to affect
ionic interactions within the solution.
[0079] Similarly, labeling species may be added following an
initial operation, where such labeling species might affect the
previous operation. One example of such labeling includes, for
example, addition of labeled antibodies to specific proteins,
thereby allowing the system to function as a chip-based western
blotting system. Specifically, following protein separation, a
labeled antibody is added to the separated proteins just prior to
detection, to preferentially associate with a protein bearing a
recognized epitope. The protein is then detected by virtue of its
size, and its ability to be recognized by a selected antibody.
[0080] F. Overall Systems
[0081] The devices and reagents of the present invention are
typically used in conjunction with an overall analytical system
that controls and monitors the operation and analyses that are
being carried out within the microfluidic devices and utilizing the
reagents described herein. In particular, the overall systems
typically include, in addition to a microfluidic device or
capillary system, an electrical controller operably coupled to the
microfluidic device or capillary element, and a detector disposed
within sensory communication of the separation zone or channel of
the device.
[0082] An example of a system according to the present invention is
shown in FIG. 2. As shown, the system 200 includes microfluidic
device 100, which comprises a channel network disposed within its
interior portion, where the channel network connects a plurality of
reservoirs or sample/reagent wells. An electrical controller 202 is
operably coupled to the microfluidic device 100 via a plurality of
electrodes 204-234 which are placed into contact with the fluids in
reservoirs of the microfluidic device 100. The electrical
controller 202 applies an appropriate electric field across the
length of the separation channel of the device to drive the
electrophoresis of the sample materials, and consequent separation
of the proteins and polypeptides of the invention. In the case of
microfluidic devices that include intersecting channel networks,
e.g., as shown, the electrical controller also applies electrical
currents for moving the different materials through the various
channels and for injecting those materials into other channels.
Electrical controllers that provide selectable current levels
through the channels of the device to control material movement are
particularly preferred for use in the present invention. Examples
of such "current controllers" are described in detail in U.S. Pat.
No. 5,800,690, which is incorporated herein by reference.
[0083] The overall system 200 also includes a detector 204 that is
disposed in sensory communication with the separation channel
portion of the channel network in the microfluidic device 100. As
used herein, the phrase "in sensory communication" refers to a
detector that is positioned to receive a particular signal from a
channel within a microfluidic device. For example, in the case of
microfluidic devices that are used to perform operations that
produce optical signals, e.g., chromophoric, fluorescent or
chemiluminescent signals, the detector is positioned adjacent to a
translucent portion of the device such that optical elements within
the detector receive these optical signals from the appropriate
portion of the microfluidic device. Electrochemical detectors, on
the other hand, in order to be in sensory communication, typically
include electrochemical sensors, e.g., electrodes, disposed within
the appropriate channel(s) of the device, so as to be able to sense
electrochemical signals that are produced or otherwise exist within
that channel. Similarly, detectors for sensing temperature will be
in thermal communication with the channels of the device, so as to
sense temperature or relative changes therein. In preferred
aspects, optical detectors are employed in the systems of the
present invention, and more preferably, optical detectors that are
configured for the detection of fluorescent signals. As such, these
detectors typically include a light source and an optical train for
directing an activation light at the separation channel, as well as
an optical train and light sensor, for collecting, transmitting and
quantifying an amount of fluorescence emitted from the separation
channel. In general, a single optical train is utilized for
transmission of both the activation light and the fluorescent
emission, relying upon differences in wavelengths of the two types
of energy to distinguish them. Generally, optical sensors
incorporated into the optical detectors of the present invention
are selected from these that are well known in the art, such as
photomultiplier tubes (PMT) photodiodes, and the like. In
particularly preferred aspects, an Agilent 2100 Bioanalyzer is used
as the controller/detector system (Agilent Technologies).
[0084] The systems described herein also typically include a
processor or computer 206 operably coupled to the electrical
controller, for instructing the operation of the electrical
controller in accordance with user instructions or preprogrammed
operating parameters. The computer is also typically operably
coupled to the detector for receiving and analyzing data that the
detector receives from the microfluidic device. Accordingly, the
computer typically includes appropriate programming for directing
the operation of the electrical controller to apply electric fields
to inject each of a potential plurality of samples into the
separation channel. Typically, the computer also is operably
coupled to the detector so as to receive the data from the detector
and to record the signals received by the detector. Processor or
computer 206 may be any of a variety of different types of
processors. Typically, the computer/processor is a IBM PC or PC
compatible computer, incorporating an microprocessor from, e.g.,
Intel or Advanced Microdevices, e.g., Pentium.TM. or K6.TM., or a
MacIntosh.TM., Imac.TM. or compatible computer.
[0085] In the case of the polypeptide characterization methods of
the present invention, the computer or processor is typically
programmed to receive signal data from the detector, and to
identify the signal peaks that correspond to a separated protein
passing the detector. Typically, one or more internal standard
proteins may be run along with the sample material. In such cases,
the computer is typically programmed to identify the standard(s)
e.g., by its location in the overall separation, either first or
last, and to determine the molecular weights of the unknown
polypeptides in the sample by extrapolation or interpolation from
the standard(s). A particularly useful computer software program
for use in accordance with the present invention is described for
use with separation methods, in Provisional Patent Application No.
60/068,980, filed Dec. 30, 1997, and incorporated herein by
reference. In the case of those embodiments run on an Agilent 2100
Bioanalyzer, the computer typically includes software programming
similar to that offered used to run these systems for nucleic acid
analysis.
[0086] G. Kits
[0087] The present invention also provides kits for use in carrying
out the described methods. Generally, such kits include a capillary
or microfluidic device as described herein. The kits also typically
include the various components of the separation buffer, e.g., the
non-crosslinked polymer sieving matrix, detergent, buffering agent
and the lipophilic dye. These components may be present in the kit
as separate volumes of preformulated buffer components, which may
or may not be pre-measured, or they may be provided as volumes of
combined preformulated reagents up to and including a single
combination of all of the reagents, whereby a user can simply place
the separation buffer directly into the microfluidic device. In
addition to the buffer components, kits according to the present
invention also optionally include other useful reagents, such as
molecular weight standards, as well as tools for use with the
devices and systems, e.g., instruments which aid in introducing
buffers, samples or other reagents into the channels of a
microfluidic device.
[0088] In the kit form, the reagents, device and instructions
detailing the use thereof are typically provided in a single
packaging unit, e.g., box or pouch, and sold together. Provision of
the reagents and devices as a kit provides the user with
ready-to-use, less expensive systems where the reagents are
provided in more convenient volumes, and have all been optimally
formulated for the desired applications, e.g., separation of high
molecular weight vs. low molecular weight proteins.
[0089] H. Automated Protein Analysis
[0090] In the previously described microfluidic devices of FIGS. 1
and 7, the protein samples being characterized need to be placed
into reservoirs on the microfluidic device. The scope of the
invention also encompasses microfluidic devices that are capable of
obtaining protein samples from sources outside the microfluidic
device. This can be accomplished by extending a sampling pipettor
or capillary from the channel network within the device into an
external sample source such as a well in a multiwell plate. The
sample in the external source can be drawn into the capillary, or
"sipper", by pressure or electrokinetic forces. Multiwell plates
come in standard formats, such as the 96, 384, or 1536 well
formats, that are compatible with a variety of commercially
available fluid-handling equipment.
[0091] FIG. 9 schematically illustrates a system 900 that comprises
a microfluidic device 902 comprising a sipper 903, a channel
network 905, and a plurality of reservoirs 906. The sipper 903 is
attached to the device 902 such that a channel within the capillary
(not shown) is in fluid communication with the channel network 905.
A multiwell plate 908 comprising a plurality of wells that act as
external sample sources is provided so as to be accessible by the
capillary element 903. This multiwell plate 908 could be a standard
format plate with protein samples placed in the wells. In many
embodiments, it may be desirable to employ a second multiwell plate
913 containing standards. For example, the wells in multiwell plate
913 could contain protein ladders comprising polypeptides of known
size. Typically, one or all of the device 902 and the multiwell
plates 908,913 are coupled to an x-y-z translation stage 909 that
moves one or all of these components relative to the other.
Typically, the x-y-z translation stage 909 is automatically
controlled, e.g., by a robotic positioning system (not shown). Such
robotic x-y-z translation systems are commercially available.
[0092] The other components of the system in FIG. 9, such as the
controller 917, the computer 918, and the detector 919, are
analogous to the system components shown in FIG. 2. The controller
917 controls the movement of fluid within the microfluidic device
902. In the embodiment of FIG. 9, the controller applies a negative
pressure to a reservoir on the microfluidic device 902 through a
pressure lumen 923. Application of the negative pressure causes
fluid to be drawn from one of the sample sources in the multiwell
plate 908 through the capillary 903 and into channel network 905.
In various embodiments, the controller could direct the application
of other positive or negative pressures, or electric fields, or a
combination of pressure and electric fields, to effectuate movement
of fluid through the channel network 905. A system in accordance
with the invention also typically includes a processor or computer
918 that interfaces with both the controller 917 and a detector
919. The computer in the embodiment of FIG. 9 also directs the
x-y-z translation stage 909. And finally, a detector 919 is also
provided within sensory communication of one or more channels in
the channel network 905. Data from the detector 919 is collected,
stored and/or analyzed by a computer or processor 918.
[0093] FIG. 10 shows an example of a microfluidic device 902 that
can be employed in the embodiment of FIG. 9. Except for the
additional process steps necessitated by the introduction of a
protein sample from an external source, a protein analysis carried
out in the microfluidic device of FIG. 10 is almost identical to
the analysis carried out in the microfluidic device in FIG. 7.
Protein samples drawn from an external source through the capillary
enter the channel network of the microfluidic device 902 at
intersection 940. In the embodiment of FIG. 10, fluid from the
external source is drawn into the sipper by applying a reduced
(i.e. below atmospheric) pressure applied to reservoir 915.
Reservoir 910 is left open to the atmosphere so that the reduced
pressure applied to reservoir 915 also induces a flow from
reservoir 910 into channel 912. The fluid in reservoir 910 mixes
with the sample as it enters the microfluidic device at the
sipper/channel intersection 940. The fluid in reservoir 910 can
comprise a diluent such as water so the concentration of the sample
can be modified. The fluid in reservoir 910 may also contain
components such as polypeptide standards (i.e. markers), or
reagents such as salts or buffering agents. The mixture of sample
and fluid from reservoir 910 then flows through intersection 942
and channels 914 and 916 toward waste reservoir 915. At least a
portion of the mixture flowing through channel 914 can be
redirected to flow through injection intersection 944 by applying
an electrical field between reservoirs 925 and 920. The magnitude
and direction of the field are configured to produce an
electrokinetic flow that directs the mixture through intersection
944 into channel 921 towards reservoir 920.
[0094] The next step in the analysis is to inject the mixture of
sample and fluid from reservoir 910 flowing through intersection
944 into separation channel 904, where the polypeptides in the
sample are separated by size. Various embodiments of the invention
may employ different injection methods. For example, as previously
described with respect to the embodiments of FIGS. 1 and 7,
pinching currents can be applied at injection intersection 944 so
that the mixture containing the protein sample does not diffuse
into the separation channel 904 before sample injection takes
place. Methods of pinching a flow and of injecting a pinched flow
are disclosed in the previously cited WO 96/04547. To illustrate
how a pinched flow may be employed in the microfluidic device of
FIG. 10, FIG. 11A shows an expanded view of intersection 944 of the
microfluidic device 902 in FIG. 10. The sample-containing mixture
(shaded portion) flowing from channel 914 into channel 921 through
intersection 944 is constrained by pinching flows (represented by
the arrows) entering the intersection 904 from both sides of
separation channel 904. Both of the pinching flows could comprise
separation buffer. To inject the mixture into the separation
channel 904, an electric field is applied across separation channel
904 so that the mixture in injection intersection 944 flows into
separation channel 904 towards waste reservoir 965. During
injection, voltages may be applied to reservoirs 920 and 925 that
pull the material in channels 914 and 921 away from injection
intersection 944 to prevent leakage of the fluid in those channels
into the separation channel 904. As the mixture travels through the
separation buffer in separation channel 904, the polypeptides in
the sample within the mixture are electrophoretically
separated.
[0095] Embodiments employing a pinched injection scheme can be
employed only when the concentration of the protein in the sample
is relatively high. There are alternative injection schemes that
can increase the sensitivity of the analysis by increasing the
amount of protein in the sample subjected to electrophoretic
separation. The amount of protein in the sample can be increased by
injecting a larger volume of sample-containing mixture into the
separation channel 904. FIG. 11B illustrates how one such an
injection scheme could be employed in the microfluidic device 902
of FIG. 10. While the mixture containing the protein sample flows
from channel 914 into channel 921 through intersection 944, the
electrical field across the length of separation channel 904 is
adjusted so that a portion of the mixture passing through the
injection intersection 944 accumulates in separation channel 904
before injection takes place. The accumulation may result from the
mixture diffusing and/or flowing into the separation channel 904.
In many embodiments, a portion of the mixture flowing across
intersection 944 will accumulate in separation channel 904 when no
voltage is applied across the length of the separation channel 904.
In other words, in some embodiments the sample-containing mixture
will accumulate in the separation channel 904 before injection if
electrodes in reservoirs 960 and 965 are allowed to float while
sample-containing mixture flows from channel 914 into channel 921
across intersection 944. In many embodiments, however, it may be
desirable to adjust the voltages applied to the electrodes in
reservoirs 960 and 965 so that sample flows into the separation
channel 904 before injection. In the embodiment of FIG. 11B, for
example, voltages are applied to reservoirs 960 and 965 to create a
flow of sample-containing mixture into the separation channel 904.
The direction of the flow into the separation channel 904 is
indicated by the arrows, while the shading schematically
illustrates how the mixture may distribute in the separation
channel 904. As will be recognized by those in the art, the amount
of sample-containing mixture that accumulates in the separation
channel can be controlled by varying the magnitude and duration of
the flow into the separation channel. Once the desired amount of
sample containing mixture has accumulated in the separation channel
904, an electric field is applied across separation channel 904 so
that the mixture in the separation channel 904 flows towards waste
reservoir 965. Just as in embodiments that employ pinched
injection, the material in channels 914 and 921 may be pulled away
from injection intersection 944 during and after injection.
[0096] As in all previously described embodiments, the polypeptides
in the sample-containing mixture injected into separation channel
904 are electrophoretically separated. The separation is performed
by creating an electric field across separation channel 904 by
applying a voltage across electrodes immersed in buffer reservoir
960 and waste reservoir 965. Buffer reservoir 960 contains
separation buffer that, as in the previously described embodiments,
typically comprises a polymer, a buffering agent, a detergent, and
a lipophilic dye. The electric field causes the polypeptides from
the sample to separate according to size as they electrophorese
through the polymer in the separation channel 904. The device 902
in FIG. 10 is configured to carry out the previously described
post-separation treatment methods. In other words, like the
embodiment of FIG. 7, the embodiment of FIG. 10 is configured to
dilute the separation buffer containing the sample components below
the CMC before that separation buffer reaches the detection region
970. The dilution takes place by flowing diluent from diluent
reservoirs 930 and 934 through diluent channels 930a and 934a into
the separation channel 904. The diluent typically comprises the
polymer and buffer components of the separation buffer. The
fluorescence peaks produced by the various electrophoretically
separated polypeptides are detected at detection region 970 by the
previously described methods.
[0097] The ability to obtain samples from external sources gives
microfluidic devices, such as the device in FIG. 10, the ability to
analyze a large number of protein samples as part of an automated
process. A commercial system is available that allow a microfluidic
device with a sipper to automatically obtain samples from a
multiwell plate. This system, the AMS 90 SE Electrophoresis System,
is manufactured and marketed by Caliper Life Sciences, Inc. of
Mountain View Calif. When a microfluidic device is used to process
a large number of protein samples, however, the performance of the
chip may degrade or drift after processing several samples. For
example, the elution time and/or area of the fluorescence peaks
(e.g., like the peaks in FIG. 8D) in two analyses of the same
protein sample may be different if more than twelve other samples
were analyzed on the same device between those two analyses. Such
variation in elution time inhibits the ability of an analysis to
identify polypeptides, while the variation in peak area inhibits
the ability of an analysis to provide quantitative measurements of
protein concentration. Another issue that arises when processing a
large number of samples is the uniformity of the pretreatment
conditions of the samples. In other words, it is desirable for the
protein analysis performed on a microfluidic device to be robust
enough so that the microfluidic device is able to process protein
samples with a variety of different salt, buffer, and detergent
concentrations.
[0098] The same reagents used in conjunction with the embodiment of
FIG. 7 can be employed for the processing of large numbers of
protein samples in the embodiments of FIGS. 9 and 10. Specifically,
the separation buffer may comprise a 10 mM to 200 mM concentration
of buffering agent, a 0.01% to 1% concentration of a detergent such
as sodium dodecyl sulfate (SDS), and a dye concentration of between
0.1 .mu.M and 1 mM. To bring the detergent concentration below the
CMC, which for SDS is around 0.1%, the separation buffer is diluted
in a range of about 1:2 to 1:30. Within these ranges of reagent
compositions, however, there are subranges that provide for a more
stable and robust protein analysis better suited for the processing
of a large number of samples. For example, the stability of a
protein analysis in accordance with the invention may be improved
if the detergent concentration in the separation buffer is between
0.05% and 0.4%, preferably between 0.1% and 0.3%, and most
preferably between 0.15% and 0.25%. The dilution ratios for the
improved process are in the range of 1:2 to 1:10, preferably 1:3 to
1:8, and more preferably in the range of 1:4 to 1:7.
[0099] The robustness of a protein analysis, i.e. the ability of
the analysis to provide quantitative measurements of samples with
varying salt and detergent concentrations, may be improved by
increasing the salt concentration in the sample-containing mixture
that is injected into the separation channel. In previously
described embodiments of the invention, the sample typically had a
non-zero salt concentration due to the use of buffering agents such
as Tris-Tricine during pretreatment. In those embodiments, the
buffer concentration in the sample-containing mixture injected into
the separation channel (e.g. 704 in FIG. 7) is typically within the
previously cited range of buffer concentrations for the separation
buffer. For many buffers, the effective ionic concentration may be
lower than the buffer concentration. For example, a buffer solution
comprising a Tris-Tricine buffer formulated from 120 mM Tricine and
40 mM Tris would have an effective ionic concentration in excess of
5 mM. Increasing the ionic concentration of the sample-containing
mixture above that level improves the stability of the protein
analysis. Increasing the ionic concentration in the
sample-containing mixture, however, also tends to reduce the
sensitivity of the analysis. In other words, increasing the ionic
concentration tends to increase the difficulty of detecting sample
components of low concentration. Accordingly, there are limits on
how high the ionic concentration should be increased. For example,
the ionic concentration of the sample-containing mixture may be
increased to between 10 mM and 1M, preferably between 50 mM and 500
mM, and more preferably between 100 mM and 500 mM. The ionic
concentration may be brought into those ranges by adding salts such
as NaCl, TrisCl, or phosphate buffer saline (PBS) to the sample
during pretreatment, or by mixing a solution containing one or more
salts to the sample before it is injected into the separation
channel. In the embodiment of FIG. 10, the salt concentration in
the sample-containing mixture could be increased by adding salt to
the sample during pretreatment, or by adding salt to the solution
in reservoir 910 that mixes with the sample that enters the
microfluidic device 902 at intersection 940. In some embodiments it
may be preferable to employ multicomponent salts such as PBS. For
example, in a protein analysis operated with reagent concentrations
in the previously described subranges of reagent concentrations
optimized for the processing of a large number of samples, the
robustness of the analysis can be further improved through the
addition of PBS to the sample in a concentration range of
0.01.times. to 10.times., preferably 0.05.times. to 5.times., and
more preferably 0.05.times. to 2.times.. Such a protein analysis
should be able to accommodate protein samples with salt
concentrations of 0M-1M, and detergent concentrations of between 1%
and 2%, with sensitivity comparable to or better than the
sensitivity of standard SDS-PAGE analyses.
[0100] While modification of the formulation of the separation
buffer and sample-containing mixture can improve the stability and
robustness of a protein analysis in accordance with the invention,
proper use of calibration standards can further improve the
analysis. One simple method of using a calibration standard in an
embodiment of the invention is to analyze a protein sample
comprising a protein ladder comprising a plurality of polypeptides
of known molecular weight before analyzing protein samples made up
of polypeptides of unknown size. The molecular weight of the
polypeptides of unknown size would be estimated by comparing the
elution times of those polypeptides to the elution times of the
molecular weight standards in the ladder. It is typically
advantageous to perform this comparison by deriving an empirical
mathematical correlation between molecular weight and elution time
for the molecular weight standard protein ladder, and then using
that correlation to calculate estimates of molecular weight base on
elution times.
[0101] The use of only a single standard protein ladder, however,
does not compensate for the drift in the process that may occur
over the course of measuring a large number of samples. In other
words, it would be advantageous to periodically recalibrate the
process results to compensate for process drift. Periodic
recalibration may comprise interspersing repeated analyses of a
single protein ladder comprising known molecular weight
polypeptides within a series of analyses of protein samples. In the
system of FIG. 9, for example, periodic recalibration could be
accomplished by placing identical standard protein ladders in the
eight wells of multiwell plate 913, and measuring those standard
ladders before and/or after each of the eight twelve-sample rows in
multiwell plate 908. When the same standard ladder is measured
before and after the twelve samples in the row, the changes in the
elution times of the polypeptides in the ladder are indicative of
process drift. A mathematical expression can be used to correct for
this process drift. In one embodiment, the correction can be
applied to the analysis of a protein sample by deriving elution
time/molecular weight correlations for two standard ladders
measured before and after that sample. A mathematical expression
for the process drift can be derived by comparing the elution time
profiles of the two standard ladders. For example, a weighted
average of the elution time/molecular weight correlations for each
standard ladder can be used to determine a elution time/molecular
weight correlation to be used for a particular protein sample. For
example, in the embodiment of FIG. 9, if a standard ladder were
measured before and after each row, which contains twelve samples,
then the analysis of the second ladder would be the thirteenth
analysis performed after the analysis of the first ladder.
Accordingly, the correlation applied to the first sample in the row
of multiwell plate could be weighted average of the first and
second ladder correlations where the first ladder correlation is
weighted by a factor of 12/13, while the second ladder correlation
is weighted by a factor of 1/13. In the analysis of the second
sample of the row, the weighting factors could be 11/13 and 2/13
for the first and second ladders respectively. Although this
illustrative weighting scheme is inherently linear, and is based on
only two standard ladder measurements, any linear or nonlinear
function can be used to determine how the elution time/molecular
weight data from the analyses of two or more standard protein
ladders can be used to derive elution time/molecular weight
correlations for the protein samples analyzed between or among
those ladders.
[0102] Further compensation for process drift can be obtained by
placing one or more markers in each protein sample. These markers,
which are polypeptides of known molecular weight, provide reference
peaks of known molecular weight. As a practical matter, the
molecular weight of the markers need to be outside the range of
molecular weights of the polypeptides in the sample so that the
markers can be identified, and so that the peaks produced by the
markers do not overlap with any sample peaks. It is often difficult
to find a marker that has a higher molecular weight than all of the
polypeptides of potential interest in a sample. Accordingly, it is
often desirable to employ only a single marker with a lower
molecular weight than all of the polypeptides of interest. In some
embodiments, the fluorescence peak produced by an unbound dye may
serve as the single lower marker. For example, the peak produced by
Alexa Fluor dye (commercially available from Molecular Probes, Inc.
of Eugene Oreg.) elutes at a time corresponding to a molecular
weight below the molecular range of interest for most analyses. If
an Alexa Fluor marker is added to each sample in a series of
protein samples to be analyzed, then the Alexa Fluor peak can
provide a standard to which the elution time of the sample
components can be compared. In some embodiments, the concentration
of the Alexa Fluor dye sample-containing mixture injected into the
separation channel is in range of 0.1 .mu.M to 10 .mu.M, preferably
0.1 .mu.M to 5 .mu.M, and more preferably 0.1 .mu.M to 10
.mu.M.
[0103] FIGS. 12A through 12C illustrate how the use of periodic
recalibration using a standard protein ladder combined with the use
of a lower marker can correct for process drift. FIG. 12A
represents the raw data from the analysis of fourteen protein
samples. Each column of bands represents the polypeptide peaks from
an analysis, where peak elution time increases from the bottom to
the top. The order of the analyses is from left to right. In other
words, the data from the first analysis are in the left-most
column, while the data from the fourteenth and last analysis are in
the right-most column. The first and fourteenth analyses were
performed on identical protein ladders, which indicated by their
data being placed within boxes. The twelve samples analyzed between
the standard ladders comprise various subsets of the polypeptides
in the ladders. In the data from every analysis, the bottom-most
(i.e. first eluting) peak corresponds to an Alexa marker placed
within each sample. FIG. 12A shows that over the course of the
fourteen measurements, the elution times of the peaks drift. FIG.
12B shows the effect of using the lower marker elution time to
correct the data in FIG. 12A. For the second through fourteenth
runs in FIG. 12B, the elution times for each peak were multiplied
by the ratio of the elution time of the Alexa peak in that run to
the elution time of the Alexa peak in the first run. The corrected
elution times produced by multiplying each peak elution time by
this ratio causes the Alexa peak in each of the second through
fourteenth runs to have the same elution time as the Alexa peak in
the first run. Thus the bottom peak for each run in FIG. 12B has
the same corrected elution time. The peaks for the other
polypeptides still reflect a component of process drift that
appears to be a function of elution time. To correct for this
drift, a linear function of process drift as a function of elution
time was generated by comparing the elution times of corresponding
peaks in the two ladder measurements (the first and fourteenth
analyses) in FIG. 12B. The application of this linear function to
the results of FIG. 12B produced the doubly corrected data in FIG.
12C. Thus the use of a single marker in each run coupled with
periodic recalibration with standard ladders can be used to
mitigate the effects of process drift.
[0104] Standards and markers may also be employed to increase the
accuracy of quantitative estimates of protein concentration. In
analyses in accordance with the invention, the area of a
fluorescence peak corresponding to a certain molecular weight
polypeptide can often be correlated to the concentration of that
polypeptide. When an identical concentration of Alexa Fluor dye is
introduced into a series of protein analyses carried out on a
microfluidic device, changes in the Alexa Fluor peak area indicate
a process drift in the protein analysis. To compensate for the
drift, the peak area in each analysis can be normalized so the
Alexa Fluor peak in each analysis is essentially the same. This
normalization procedure is capable of improving the consistency of
the quantitative results produced by a series of protein analyses.
In other embodiments, the peaks areas produced by a protein ladder
comprising polypeptides of known molecular weight and known
concentration may be used to monitor process drift. By examining
peak areas of polypeptides of different molecular weights, any
changes in peak area that are a function of molecular weight or
elution time can be compensated for. Mathematical techniques
analogous to those used to correct for the effects of process drift
on elution times can also be used to correct for the effect of
process drift on peak area.
[0105] The present invention is further illustrated with reference
to the following examples that demonstrate certain aspects of the
invention without limiting the scope of that invention.
EXAMPLES
[0106] All experiments were performed in a twelve-sample
microfluidic device having a single separation channel and the
channel geometry illustrated in FIG. 1. Control and detection were
performed using a multichannel, twelve electrode electrical
controller/detector having a single point laser fluorescence
detector located along the single separation channel.
Example 1
Separation of Polypeptides Using SubCMC Separation Buffer
[0107] Fluorescence data received from the separation channel was
recorded by a computer (PC with Intel Pentium.RTM. microprocessor).
The data was displayed in both a linear plot of fluorescence vs.
time as well as in an emulated gel format generated by Caliper
Technologies Corp. proprietary software.
[0108] A 0.5 M solution of Tris-Tricine buffer was prepared by
dissolving Tricine in deionized water at a 0.5 M concentration, and
adjusting the pH to 7.5 with 1 M Tris. The resulting buffer was
then filtered through a 0.22 .mu.m syringe filter. The sieving or
separation buffer was prepared at 3%
polydimethylacrylamide-coacrylic acid in 12.5 mM Tris-Tricine
buffer with 0.9% (w/v) sodium dodecyl sulfate (SDS), and 10 .mu.M
Syto 60 dye (Molecular Probes, Eugene Oreg.). The separation buffer
was then filtered through a Costar Spin-X.TM. 0.22 .mu.m cellulose
acetate centrifuge filter.
[0109] Samples were pretreated in denaturation buffer prior to
placement into the reservoirs of the device. The denaturation
buffer was 0.75% SDS (w/v) and 1% 2-mercaptoethanol (v/v)(BME) in
250 mM Tris-Tricine buffer. The samples were mixed 1:1 with
denaturation buffer (e.g., 20 .mu.l sample and 20 .mu.l buffer) in
a 0.5 ml microfuge tube and heated to 100.degree. C. for 10
minutes. The heated samples were then centrifuged and vortexed.
Prior to loading the samples into the wells of the microfluidic
device, they were diluted 1:10 with deionized water, e.g., 1 .mu.l
sample/buffer and 9 .mu.l water). The prepared samples therefore
had a detergent concentration of 0.0375% SDS.
[0110] To prepare the microfluidic device, 7.5 .mu.l of separation
buffer was pipetted into well 166 of a clean, dry device, and
pressurized with a syringe to force the separation buffer into all
of the channels of the device. 7.5 .mu.l of separation buffer was
then pipetted into each of wells 164, 168 and 170. 0.5 .mu.l of the
diluted samples were then separately pipetted into each of wells
140-162. In the example shown in FIG. 4, standards of known
molecular weight were used. The standards included ovalbumin (45
kD), bovine carbonic anhydrase (29 kD), soybean trypsin inhibitor
(21.5 kD) and .alpha.-lactalbumin (14.4 kD).
[0111] With reference to FIG. 1, wells 142 and 146 contained only
buffer, and were used as blanks. A standard protein solution
containing 100 .mu.g/ml of each of the four protein standards was
placed into each of wells 150 and 154, while a solution of the same
four proteins at 500 .mu.g/ml was placed into wells 158 and 162. A
solution containing just the carbonic anhydrase standard at 1000
.mu.g/ml was placed into wells 140 and 144. A solution containing
both carbonic anhydrase and trypsin inhibitor at 100 .mu.g/ml, was
placed into wells 148 and 152, while a solution containing the same
proteins, but at 500 .mu.g/ml was placed into wells 156 and
160.
[0112] Each sample was separately injected down the main separation
channel 104 and the separated components were detected as a
function of retention time from injection. The chromatogram for
each run was displayed in the form of dark bands intended to
emulate a standard coomassie stained SDS-PAGE gel. Each lane of the
emulated gel represents a chromatogram for a separate sample, with
the dark bands indicating increases in fluorescence over
background. In particular, a mixture of ovalbumin (45 kD), bovine
carbonic anhydrase (29 kD), soybean trypsin inhibitor (21.5 kD) and
.alpha.-lactalbumin (14.4 kD) was prepared. The two different
concentrations of the four protein mix were run at 100 .mu.g/ml
(Lane A2, well 154) and 500 .mu.g/ml (Lane A3, well 162). Separate
mixtures of each of these standards were also prepared and run as
follows:
1 Lane B1 (well 144): Carbonic Anhydrase (1 mg/ml) Lane B2 (well
152): Trypsin Inhibitor and Carbonic anhydrase (both at
100/.mu.g/ml) Lane B3 (well 160): Same as B2 (both at 500 .mu.g/ml)
Lane C2 (well 142): Same as Lane A2 Lane C3 (well 150): Same as
Lane A3 Lane D1-D3 (wells 140-156): Same as B1-B3
[0113] FIG. 5 shows a plot of the log of the molecular weight
versus the migration time for a set of standards run in the same
fashion as described above. As can be seen, the separation methods
described yield accurate, e.g., linear data, which permits the
characterization of proteins of unknown molecular weight, by
correlating the migration times for those unknown proteins with the
set of standards, in accordance with the plot shown. As can be seen
from FIGS. 4 and 5, a highly reproducible, accurate and rapid
method is provided for characterizing proteins and other
polypeptides.
[0114] The same set of standards, also including a Cy-5 dye marker
was also run to show the co-elution of the detergent dye front. The
chromatogram from this run is shown in FIG. 6. As can be seen, the
detergent-dye peak (indicated with an asterisk) elutes at
substantially the same time as proteins having a molecular weight
of in the range of 65 kD. In those instances where the detergent
concentration in the sample pretreatment buffer is at levels
previously described in the art, e.g., 2%, the indicated peak is
much larger, and that peak substantially interferes with the
identification and quantitation of proteins in this molecular
weight range.
Example 2
Separation and Detection of Polypeptides Using
Post-Separation/Pre-Detecti- on Dilution
[0115] A microfluidic device as shown in FIG. 7, was filled with a
separation buffer as described above. The separation channel 704 is
intersected by the diluent channels 720a and 722a at point 1.2 cm
downstream from the injection point, and 0.1 cm upstream of the
detection point 732. The separation buffer contained 4.2%
non-crosslinked polydimethylacrylamide/co-acrylic acid in 30 mM
Tris Tricine buffer, and 0.13% SDS. The dilution buffer, which
comprised 30 mM Tris-Tricine with no polymer or SDS, was placed
into reservoirs 720 and 722. The buffering agent was flowed into
the separation channel electrokinetically, e.g.,
electrophoretically.
[0116] A polypeptide standard solution (10-205 kD protein standard
from Bio-Rad, Inc.) was placed into a sample reservoir, e.g.,
reservoir 706, and loaded and injected into the separation channel
using the same methods described in U.S. Pat. No. 5,976,336,
previously incorporated herein.
[0117] FIGS. 8A-8D illustrates plots of fluorescence versus time,
as detected at the detection point 732 in a 2100 Bioanalyzer
(Agilent Technologies, Inc.) for a standard separation performed
without a post separation treatment and with a post separation
dilution. Specifically,
[0118] FIGS. 8A and B show a blank run (no polypeptides in the
sample) and a protein sample run in a microfluidic device having no
post separation dilution functionality. The device was functionally
similar to the device channel layout shown in FIG. 1. As shown, the
data from the blank and polypeptide runs included substantial
background and other baseline problems including a large detergent
dye front, followed by a baseline divot and a following dye hump.
These same baseline deviations were found in the sample separation
run, which cause substantial difficulty in qualifying and
quantifying the separation data. FIGS. 8C and 8D illustrate the
same blank run and polypeptide sample analysis using a post
separation dilution step where the Tris Tricine buffer was
introduced into the separation channel downstream of the majority
of the separation, but upstream of the detection point. As shown,
the post-separation dilution step substantially reduces overall
background fluorescence relative to the detected sample components
over the non-diluted samples, while also reducing the baseline
humps and dips that are associated with micelle dye binding, e.g.,
as seen in FIGS. 8A and 8B.
[0119] Unless otherwise specifically noted, all concentration
values provided herein refer to the concentration of a given
component as that component was added to a mixture or solution
independent of any conversion, dissociation, reaction of that
component to a alter the component or transform that component into
one or more different species once added to the mixture or
solution.
[0120] All publications and patent applications are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference. Although the present
invention has been described in some detail by way of illustration
and example for purposes of clarity and understanding, it will be
apparent that certain changes and modifications may be practiced
within the scope of the appended claims.
* * * * *